Strategies for production of proteins in heterologous fusion form have been widely applied in biotechnology for many purposes, such as secretion of proteins from host cells (fused to signal peptides), easy detection or purification of protein products (fused to reporter enzymes for detection and to peptide tags for purification), searching for proteins with desired biological activities (e.g., in the phage display technique and the two-hybrid system). Enhanced expression of proteins of interest has also been achieved by N-terminal fusion of a small peptide to the target protein. Fusion of a ubiquitin gene together with a ubiquitin promoter to the 5′-end of a gene of interest is one of the systems which has been used to enhance protein expression. Ubiquitin exists in all eukaryotic cells and is the most highly conserved protein yet identified. It is abundant in cells and exhibits profound stability to heat and proteolytic degradation. Moreover, ubiquitin precursors, that is, polyubiquitin where ubiquitin monomers are linked up head to tail and ubiquitin extension proteins where a single ubiquitin is appended at its C-terminus to either of two small ribosomal proteins, undergo rapid processing by ubiquitin C-terminal hydrolases, which cleave C-terminal of the ubiquitin moieties and release the free ubiquitin monomer and the C-terminal extension proteins. All of these features have rendered ubiquitin as an excellent N-terminal fusion partner to augment target protein accumulation in genetic engineering.
The ubiquitin fusion approach was first developed by Butt et al. (1989), who showed that fusion of ubiquitin to yeast metallothionein or to the α subunit of the adenoylate cyclase-stimulatory GTP-binding protein increased the yield of these otherwise unstable or poorly expressed proteins from undetectable levels to 20% of the total cellular proteins in E. coli. Ecker et al. (1989) demonstrated that in yeast, ubiquitin fusion resulted in enhanced expression of three mammalian proteins by up to 200-fold and all these ubiquitin fusion proteins were correctly processed by yeast ubiquitin-specific endopeptidase to release authentic functional proteins. A similar yeast ubiquitin fusion expression system was reported by Sabin et al. (1989), in which ubiquitin/human γ-interferon and ubiquitin/αl-proteinase inhibitor were highly expressed and quantitatively cleaved to yield γ-IFN and α1-PI with authentic amino termini.
Since these early reports, a wealth of studies on ubiquitin fusion expression of various proteins in E. coli and yeast have been described (Baker et al., 1994; Barr et al., 1991; Coggan et al., 1995; Gali and Board, 1995; Gehring et al., 1995; Han et al., 1994; Kiefer et al., 1992; Lu et al., 1990; Lyttle et al., 1992; Mak et al., 1989; McDonnell et al., 1989; McDonnell et al., 1991; Pilon et al., 1996; Poletti et al., 1992; Rian et al., 1993; Tan and Board, 1996; Welch et al., 1995). Very often fusion to ubiquitin led to dramatic enhancement in yield of the fusion protein in bacteria, or of the cleaved product in yeast.
Enhanced expression of foreign proteins by ubiquitin fusion has also been observed in plants. In analysis of the promoter of the tobacco polyubiquitin gene, Ubi.U4, by driving transient expression of the GUS reporter in tobacco protoplasts, Genschik et al. (1994) found deletion of the intron sequence from the Ubi.U4 fragment spanning from −263 to the end of the first ubiquitin-coding unit had no detectable influence on the GUS activity, but further deletion of the ubiquitin-coding sequence diminished the GUS activity by 55%.
None of these studies has shown the direct enhancing function of the ubiquitin fusion from a heterologous promoter. Garbarino and Belknap (1994) observed that fusion of the promoter plus ubiquitin-coding region of the potato ubiquitin extension protein gene ubi 3 to the GUS reporter gene resulted in GUS activity 5- to 10-fold higher than the direct fusion of the ubi 3 promoter to the GUS gene did in transgenic potato. Again, the synergistic effect of the ubi 3 promoter and the ubiquitin-coding sequence on the enhanced GUS activity was not excluded. In another study with a potato polyubiquitin gene, ubi 7, the same group (Garbarino et al., 1995) demonstrated that in transgenic potato plants GUS expression level from the fusion construct containing the ubi 7 promoter-5′ untranslated sequence-intron-first ubiquitin coding unit was 10 times higher than that derived by only the ubi 7 promoter with the 5′ untranslated sequence. However, the effects of the intron and the ubiquitin protein fusion in increasing expression level of the GUS reporter were not clearly discriminated.
In addition to the above mentioned journal papers, a number of patents related to the ubiquitin fusion technology have been filed since 1989. They are shown in Table 1. The publications and other materials used herein to illuminate the background of the invention or provide additional details respecting the practice, are incorporated by reference, and for convenience are respectively grouped in the appended List of References.
TABLE 1Patents related to the ubiquitin fusion technologyHostTitleInventorPat. No.Filing DatecellsGenerating desired amino-MITWO 8909829Oct. 19, 1989terminal residue in proteinRegulation metabolic stabilityMITUS 5093242Mar. 3, 1992mammal,of a proteinyeastNucleic acid constructs, malariaChironWO 9208795May 29, 1992yeastpolypeptides and vaccinesProduction of a protein with aMITUS 5196321Mar. 23, 1993E. colipredetermined amino-terminalamino acid residueYeast expression system forAmericanEP 608532Aug. 3, 1994yeastretinoid-X receptorCyanamidRecombinant DNA vectorsMascarenhasWO 9423040Oct. 13, 1994E. coliNew heat-inducible N-degronVarshavsky,WO 9521269Aug. 10, 1995protein and nucleic acidDohmen,encoding itJohnston,WuFusion proteins containing theVarshavsky,WO 9529195Nov. 2, 1995N-or C-terminal of ubiquitinJohnstonNew fusion protein of ubiquitinCarbarino,WO 9603519Feb. 8, 1996plantplant and lytic peptideJaynes,BelknapProduction of tissue factorInnis,WO 9604377Feb. 15, 1996yeastpathway-inhibitor in yeast cellsCreaseyStable recombinant ubiquitin-J. JaynesWO 9603522Feb. 8, 1996plantlytic peptide fusion proteinFusion protein encoded by aBachmair,US 5496721May 3, 1990mammal,gene constructFinley,yeastVarshavsky